LOCUS       62056_16        6694 bp DNA     circular SYN 17-NOV-2020
DEFINITION  Cloning vector (145)_sp(A)-mRuby3-P_bi, complete sequence.
ACCESSION   MT891327
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6694)
  AUTHORS   Frei T, Cella F, Tedeschi F, Gutierrez J, Stan GB, Khammash M, 
            Siciliano V.
  TITLE     Characterization and mitigation of gene expression burden in 
            mammalian cells
  JOURNAL   Nat Commun 11 (1), 4641 (2020)
  PUBMED    32934213
REFERENCE   2  (bases 1 to 6694)
  AUTHORS   Frei T, Cella F, Tedeschi F, Gutierrez J, Stan G-B., Khammash M, 
            Siciliano V.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-AUG-2020) Synthetic and Systems Biology for 
            Biomedicine, Italian Institute of Technology-IIT, Largo Barsanti e 
            Martteucci 53, Naples, Campania 80125, Italy
REFERENCE   3  (bases 1 to 6694)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun"; date: "2020"; volume: "11"; issue: "1"; pages: "4641"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-AUG-2020) Synthetic and Systems Biology for Biomedicine, Italian
            Institute of Technology-IIT, Largo Barsanti e Martteucci 53, Naples,
            Campania 80125, Italy"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..266
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     polyA_signal    283..507
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    complement(525..573)
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     CDS             complement(601..1311)
                     /label=mRuby3
                     /note="monomeric red fluorescent protein derived from
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
     promoter        complement(1345..1732)
                     /label=bidirectional TRE promoter
                     /note="Tet-responsive bidirectional promoter PTight-BI, 
                     consisting of seven tet operator sequences flanked on each 
                     side by the minimal CMV promoter"
     CDS             1769..2713
                     /label=ORF frame 1
                     /note="bright monomeric near-infrared fluorescent protein 
                     engineered from a bacterial phytochrome, with an emission 
                     maximum of 670 nm (Shcherbakova et al., 2016)"
     promoter        complement(2746..2764)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    2925..3046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3136..3425
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     rep_origin      complement(3504..4092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4266..5123)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5124..5228)
                     /label=AmpR promoter
     promoter        5298..5614
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5711..6307
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     regulatory      6540..6621
                     /label=SV40 poly(A) signal
                     /note="SV40 poly(A) signal"
                     /regulatory_class="polyA_signal_sequence"
ORIGIN
        1 gtcttctcca tcggatcctg atcaggggag ctcacgggga cagccccccc ccaaagcccc
       61 cagggatgta attacgtccc tcccccgcta gggggcagca gcgaccgccc ggggctccgc
      121 tccggtccgg cgctcccccc gcatcccgag ccggcagcgt gcggggacag cccgggcacg
      181 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
      241 acacctgggg gatacgggga aaaccgcggc atatggcctc gactgtgcct tctagttgcc
      301 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      361 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
      421 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
      481 atgctgggga tgcggtgggc tctatggctt ctgagggcta gcatcacaca aaaaaccaac
      541 acacagatct aatgaaaata aagatctttt attatcgatt taattaatcc ggagtcatta
      601 cttgtacagc tcgtccatgc caccaccaag gttgctgtat ttggcaactg ccacttctct
      661 ttgcactacg taggtttcat tgtcactctc ctcgatcctt tccaggcggt gatcaacggc
      721 atggacaccg ggcatcttga tgttcccgac ggtctttttt gacctgtaag ttgtcacgaa
      781 gttgcagtgc agatggccac caccatcaac tttcagtgcg atgtcagtgt atcctctcag
      841 accaccatct gctggataca tcatctctgt attaggctcc caacccttgg tcttcttctg
      901 catcacggga ccattggagg gaaagtttac ccctctgacc ttgacgttgt agacgagctc
      961 gccatcctca aggctggtgt cctgcgtgac ggtgacgact ccaccatctt cgtatctcgt
     1021 aactctttcc caagtaaaac cctcaggaaa ggactgttta aagaaatcag ggatgtcggc
     1081 cgggtacttg ataaaggtac ggctgccata catgaacgac gtggcaagaa tgtcaaaggc
     1141 aaatggcagg ggtcctccct cgatgacttt gatcctcatg gtttgcactc cctcgtacgg
     1201 tctgccttct ccttcacctg tgcatttgaa ttggtggccg ttgaccgaac cttccatgac
     1261 caccttcata cgcatatttt ccttgatcag ctcttcgccc ttagacacca tggtggcgcg
     1321 gccgcaccga gctcgaaatc tccaggcgat ctgacggttc actaaacgag ctctgcttat
     1381 ataggcctcc caccgtacac gcctacctcg acatacgttc tctatcactg atagggagta
     1441 aactcgacat acgttctcta tcactgatag ggataaactc gacatacgtt ctctatcact
     1501 gatagggagt aaactcgaca tacgttctct atcactgata gggagtaaac tcgacatacg
     1561 ttctctatca ctgataggga gtaaactcga catcgttctc tatcactgat agggagtaaa
     1621 ctcgacatac gttctctatc actgataggg agtaaactcg agtatgtcga ggtggcgtgt
     1681 acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagaat
     1741 tcttcgagct cggtgcgatc gcgccaccat ggtagcaggt catgcctctg gcagccccgc
     1801 attcgggacc gcctctcatt cgaattgcga acatgaagag atccacctcg ccggctcgat
     1861 ccagccgcat ggcgcgcttc tggtcgtcag cgaacatgat catcgcgtca tccaggccag
     1921 cgccaacgcc gcggaatttc tgaatctcgg aagcgtactc ggcgttccgc tcgccgagat
     1981 cgacggcgat ctgttgatca agatcctgcc gcatctcgat cccaccgccg aaggcatgcc
     2041 ggtcgcggtg cgctgccgga tcggcaatcc ctctacggag tactgcggtc tgatgcatcg
     2101 gcctccggaa ggcgggctga tcatcgaact cgaacgtgcc ggcccgtcga tcgatctgtc
     2161 aggcacgctg gcgccggcgc tggagcggat ccgcacggcg ggttcactgc gcgcgctgtg
     2221 cgatgacacc gtgctgctgt ttcagcagtg caccggctac gaccgggtga tggtgtatcg
     2281 tttcgatgag caaggccacg gcctggtatt ctccgagtgc catgtgcctg ggctcgaatc
     2341 ctatttcggc aaccgctatc cgtcgtcgac tgtcccgcag atggcgcggc agctgtacgt
     2401 gcggcagcgc gtccgcgtgc tggtcgacgt cacctatcag ccggtgccgc tggagccgcg
     2461 gctgtcgccg ctgaccgggc gcgatctcga catgtcgggc tgcttcctgc gctcgatgtc
     2521 gccgtgccat ctgcagttcc tgaaggacat gggcgtgcgc gccaccctgg cggtgtcgct
     2581 ggtggtcggc ggcaagctgt ggggcctggt tgtctgtcac cattatctgc cgcgcttcat
     2641 ccgtttcgag ctgcgggcga tctgcaaacg gctcgccgaa aggatcgcga cgcggatcac
     2701 cgcgcttgag agctaatgat agggcgcgcc tctagagggc cctattctat agtgtcacct
     2761 aaatgctaga gctcgctgat cagtccatag tcgacggatc caccggatct agataactga
     2821 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2881 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2941 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3001 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     3061 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3121 caataggccg aaatcgggga gctcacgggg acagcccccc cccaaagccc ccagggatgt
     3181 aattacgtcc ctcccccgct agggggcagc agcgaccgcc cggggctccg ctccggtccg
     3241 gcgctccccc cgcatcccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
     3301 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
     3361 ggatacgggg aaaatgatca agatcttcta cactgggacg caaggattat cctcgaggcg
     3421 cgcgaagaat caggggataa cgcagtgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3481 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3541 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3601 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3661 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3721 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3781 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3841 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3901 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3961 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4021 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4081 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4141 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4201 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4261 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4321 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4381 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4441 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4501 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4561 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4621 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4681 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4741 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4801 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4861 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     4921 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4981 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5041 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5101 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5161 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5221 gttccgcgca catttccccg aaaagtgcca cctggaggtt ttcaccgtca tcaccgaaac
     5281 gcgcgagacg aaagattggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5341 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     5401 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5461 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5521 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5581 aggaggcttt tttggaggcc taggcttttg caaaaagcta gcttgcatgc ctgcaggtcg
     5641 gccgccacga ccggtgccgc caccatcccc tgacccacgc ccctgacccc tcacaaggag
     5701 acgaccttcc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     5761 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     5821 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     5881 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     5941 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     6001 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     6061 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     6121 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     6181 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     6241 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     6301 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     6361 tggctccgac cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc
     6421 accgactcta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     6481 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     6541 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     6601 ataaagcatt tttttcactg caatctcgtg atacgcctat ttttataggt taatgtcatg
     6661 gatgccgcat agttaagcca gtatctgctc cctg
//
